EP1991567A2 - Polynucleotide encoding a maize herbicide resistance gene and methods for use - Google Patents
Polynucleotide encoding a maize herbicide resistance gene and methods for useInfo
- Publication number
- EP1991567A2 EP1991567A2 EP07752767A EP07752767A EP1991567A2 EP 1991567 A2 EP1991567 A2 EP 1991567A2 EP 07752767 A EP07752767 A EP 07752767A EP 07752767 A EP07752767 A EP 07752767A EP 1991567 A2 EP1991567 A2 EP 1991567A2
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- EP
- European Patent Office
- Prior art keywords
- class
- plant
- herbicides
- inhibiting
- herbicide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
- C12N15/8278—Sulfonylurea
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
Definitions
- This invention relates to compositions and methods useful in creating or enhancing herbicide resistance in plants. Additionally, the invention relates to plants that have been genetically transformed with the compositions of the invention.
- weeds unwanted plants
- An ideal treatment would be one which could be applied to an entire field but which would eliminate only the unwanted plants while leaving the crop plants unharmed.
- One such treatment system involves the use of crop plants that are tolerant to a herbicide. When the herbicide is sprayed on a field of herbicide-tolerant crop plants, the crop plants continue to thrive while non-herbicide-tolerant weeds are killed or severely damaged.
- Crop tolerance to specific herbicides can be conferred by engineering genes into crops which encode appropriate herbicide metabolizing enzymes. In some cases these enzymes, and the nucleic acids that encode them, originate in a plant. In other cases, they are derived from other organisms, such as microbes. See, e.g., Padgette et at. (1996) "New weed control opportunities: Development of soybeans with a Round UP ReadyTM gene” and Vasil (1996) “Phosphinothricin- resistant crops,” both in Herbicide-Resistant Crops, ed. Duke (CRC Press, Boca Raton, Florida) pp.54-84 and pp. 85-91.
- transgenic plants have been engineered to express a variety of herbicide tolerance genes from a variety of organisms, including a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et ai (1994) Plant Physiol. 106: 17), among other plant P450 genes (see, for example, Didierjean, L. er a/. (2002) Plant Physiol. 130: 179-189; Morant, M.S. et ai. (2003) Opinion in Biotechnology 14:151-162).
- AHAS acetohydroxy acid synthase
- Other genes that confer tolerance to herbicides include: acetohydroxy acid synthase ("AHAS"), which has been found to confer resistance to multiple types of ALS herbicides on plants expressing it and has been introduced into a variety of plants (see, e.g., Hattori et al. (1995) MoI. Gen. Genet. 246: 419); glutathione reductase and superoxide dismutase (Aono et al. (1995) Plant Cell Physiol. 36: 1687); and genes for various phosphotransferases (Datta et al. (1992) Plant MoI. Biol. 20: 619).
- AHAS acetohydroxy acid synthase
- herbicide-tolerant crop plants are presently commercially available, improvements in every aspect of crop production are continuously in demand.
- Herbicides and crops that are presently commercially available unfortunately have particular characteristics which can limit their usefulness in commercial crop production. Particularly, individual herbicides have different and incomplete spectra of activity against common weed species.
- acetolactate synthase or ALS (also known as AHAS) family of herbicides control weeds by inhibiting the production of branch chain of amino acids that are essential to plant growth and development. Specifically, they bind to the plant ALS enzyme. Commonly used herbicides in this family include nicosulfuron, rimsulfuron, and chlorsulfuron, among others. Herbicides in this category can be quite crop-specific.
- Embodiments of the invention relate to plants that are resistant to members of the ALS-inhibiting class of herbicides, which encompasses 5 sub-classes of herbicides including, but not limited to, the sulfonylurea (SU) family of herbicides and the imidazolinone family of herbicides.
- SU sulfonylurea
- the pigment synthesis-inhibiting class of herbicides targets the enzymes that allow plants to synthesize pigments, such as carotenoid pigments or chlorophyll pigments. Loss of pigment results in photo-destruction of chlorophyll and whitening of plant tissues, which is why these herbicides are often called "bleaching" herbicides.
- An example of a sub-class of the bleaching herbicides is the HPPD-inhibiting class, which inhibits the 4-hydroxyphenylpyruvate dioxygenase (HPPD) enzyme (Lee et al. (1997) Weed Sci. 45:601-609).
- the protoporphyrinogen oxidase (PPO)-inhibiting class of herbicides interferes with the synthesis of chlorophyll, resulting in compounds that produce highly active compounds (free-radicals). These reactive compounds disrupt cell membranes which results in the leaf burning associated with post-emergence applications of these products.
- Herbicides in this family include, but are not limited to, acifluorfen, fomesafen, lactofen, sulfentrazone, carfentrazone, flumiclorac and flumioxazin, among others.
- Embodiments of the invention relate to plants that are resistant to members of the PPO-inhibiting class of herbicides.
- Photosystem Il (PSII)-inhibiting herbicides have a mode of action that involves interaction with components in the electron transfer chain of Photosystem II. Photosynthesis requires the transfer of electrons from Photosystem Il to Photosystem I. A key step in this electron transfer chain is the reduction of plastoquinone (PQ) by the D 1 protein in the thylakoid membrane. PSII-inhibitor herbicides bind to the Di protein, thus inhibiting PQ binding and interrupting the electron transfer process. This results in the plants not being able to fix carbon dioxide and produce the carbohydrates needed for the plant to survive. The block in electron transfer also causes an oxidative stress and the generation of radicals which cause rapid cellular damage.
- PQ plastoquinone
- PSII-inhibiting herbicides are represented by several herbicide families, including the symmetrical triazines, triazinones (asymmetrical triazines), substituted ureas, uracils, pyridazinones, phenyl carbamates, nitriles, benzothiadiazoles, phenyl pyridazines.and acid amides.
- Embodiments of the invention relate to plants that are resistant to members of the PS ll-inhibiting class of herbicides.
- Synthetic auxin herbicides are a widely used class of herbicides that mimic the natural auxin hormones produced by plants. Auxins regulate many plant processes, including cell growth and differentiation. Auxins are generally present at low concentrations in the plant. Synthetic auxin herbicides mimic natural auxins and cause relatively high concentrations in the cell that result in a rapid growth response. Susceptible plants treated with these herbicides exhibit symptoms that could be called 'auxin overdose', and eventually die as a result of increased rates of disorganized and uncontrolled growth. Embodiments of the invention relate to plants that are resistant to members of the synthetic auxin class of herbicides. Some embodiments of this invention are based on the fine mapping, cloning and characterization of the gene responsible for an important herbicide resistance mechanism in maize.
- the Nsf1 resistance gene of the embodiments of the present invention encodes a novel gene related to the cytochrome P450 family. While multiple cytochrome P450 genes have been described, they differ widely in their response to different pathogens and exact action.
- the novel cytochrome P450 gene described in this disclosure has been demonstrated to provide improved tolerance or resistance to numerous herbicides, including nicosulfuron, rimsulfuron, primisulfuron, thifensulfuron and mesotrione. Summary of the Invention
- the present invention is directed to embodiments including an isolated polynucleotide comprising a nucleotide sequence encoding a polypeptide capable of conferring resistance to at least one herbicide, wherein the polypeptide has an amino acid sequence of at least 85, 90 or 95% identity, when compared to SEQ ID NO:1 based on the Needleman-Wunsch alignment algorithm, or a complement of the nucleotide sequence, wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.
- the herbicides to which the polynucleotide of the embodiments imparts resistance include members of the ALS-inhibiting class; the pigment synthesis-inhibiting class; the PPO-inhibiting class; the PS ll-inhibiting class; and the synthetic auxin class of herbicides.
- the polynucleotide of the embodiments may impart resistance to one or more herbicides from the same class, or from different classes, including representative members from all 5 classes.
- Additional embodiments of the present invention include a vector comprising the polynucleotide of the embodiments and a recombinant DNA construct comprising the polynucleotide of the embodiments, operably linked to at least one regulatory sequence.
- a plant cell, as well as a plant and a seed each comprising the recombinant DNA construct of an embodiment of the present invention are also encompassed.
- the methods embodied by the present invention include 1) a method for transforming a cell, comprising transforming a cell with the polynucleotide of an embodiment of the present invention, 2) a method for producing a plant comprising transforming a plant cell with the recombinant DNA construct of an embodiment of the present invention and regenerating a plant from the transformed plant cell, and 3) methods of conferring or enhancing resistance to at least one herbicide, comprising transforming a plant with the recombinant DNA construct of an embodiment of the present invention, thereby conferring or enhancing resistance to at least one herbicide, such as a member of the ALS- inhibiting class; the pigment synthesis-inhibiting class; the PPO-inhibiting class; the PS H-inhibiting class; and the synthetic auxin class of herbicides.
- an embodiment of the invention is a variant allele of the Nsf1 sequence in which a specific single amino acid change (see Example 2) renders the gene inoperative, resulting in sensitivity to at least one ALS or HPPD inhibitor herbicide to which most corn is resistant.
- an additional method embodied by the present invention is a method of using the variant of the Nsf1 gene as a marker in breeding strategies to avoid incorporating the sensitive allele.
- Methods of altering the level of expression of a protein capable of conferring resistance to at least one herbicide in a plant cell comprising (a) transforming a plant cell with the recombinant DNA construct of an embodiment of the present invention and (b) growing the transformed plant cell under conditions that are suitable for expression of the recombinant DNA construct wherein expression of the recombinant DNA construct results in production of altered levels of a protein capable of conferring resistance to at least one herbicide in the transformed host are also embodied by the present invention.
- optimally culture medium represents less than about 30%, about 20%, about 10%, about 5%, or about 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
- a fragment of a nucleotide sequence that encodes a biologically active portion of a polypeptide of the embodiments will encode at least about 15, about 25, about 30, about 40, or about 50 contiguous amino acids, or up to the total number of amino acids present in a full-length polypeptide of the embodiments (for example, 521 amino acids for SEQ ID NO: 2). Fragments of a nucleotide sequence that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a protein.
- an entire polynucleotide disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding polynucleotides and messenger RNAs.
- probes include sequences that are unique and are optimally at least about 10 nucleotides in length, at least about 15 nucleotides in length, or at least about 20 nucleotides in length.
- Such probes may be used to amplify corresponding polynucleotides from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism.
- Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) supra.
- comparison window makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides.
- the comparison window is at least about 20 contiguous nucleotides in length, and optionally can be about 30, about 40, about 50, about 100, or longer.
- Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, and are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the default parameters.
- CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al.
- Gapped BLAST in BLAST 2.0
- PSI-BLAST in BLAST 2.0
- PSI-BLAST in BLAST 2.0
- Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non- conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
- polynucleotide is not intended to limit the embodiments to polynucleotides comprising DNA.
- polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues.
- the polynucleotides of the embodiments also encompass all forms of sequences including, and not limited to, single-stranded forms, double-stranded forms, and the like.
- a vector is used then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
- a plasmid vector can be used.
- the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleic acid fragments of the invention. Screening to obtain lines displaying the desired expression level and pattern of the polynucleotides or of the Nsf1 locus may be accomplished by amplification, Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, phenotypic analysis, and the like.
- DNA construct refers to a DNA construct assembled from nucleic acid fragments obtained from different sources. The types and origins of the nucleic acid fragments may be very diverse.
- DNA constructs comprising a promoter operably linked to a heterologous nucleotide sequence of the embodiments are further provided.
- the DNA constructs of the embodiments find use in generating transformed plants, plant cells, and microorganisms and in practicing the methods for inducing ALS and HPPD inhibitor herbicide resistance disclosed herein.
- the DNA construct will include 5' and 3' regulatory sequences operably linked to a polynucleotide of the embodiments. Operably linked" is intended to mean a functional linkage between two or more elements.
- regulatory sequences refer to nucleotides located upstream (5 1 non-coding sequences), within, or downstream ⁇ 3' non-coding sequences) of a coding sequence, and which may influence the transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences may include, and are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.
- an operable linkage between a polynucleotide of interest and a regulatory sequence is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous.
- the coding sequence may additionally contain a sequence used to target the protein to the chloroplast , the vacuole, the endoplasmic reticulum or to the outside of the cell.
- the cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs. Such a DNA construct is provided with a plurality of restriction sites and/or recombination sites for insertion of the polynucleotide that encodes a herbicide resistance polypeptide to be under the transcriptional regulation of the regulatory regions.
- the DNA construct may additionally contain selectable marker genes.
- the DNA construct will include in the 5'-3' direction of transcription, a transcriptional initiation region (i.e., a promoter), translational initiation region, a polynucleotide of the embodiments, a translational termination region and, optionally, a transcriptional termination region functional in the host organism.
- the regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the polynucleotide of the embodiments may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the polynucleotide of the embodiments may be heterologous to the host cell or to each other.
- heterologous in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
- a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
- promoters can be used in the practice of the embodiments, including the native promoter of the polynucleotide sequence of interest. The promoters can be selected based on the desired outcome. A wide range of plant promoters are discussed in the recent review of Potenza et al. (2004) In Vitro Cell Dei/ Biol- Plant 40:1-22, herein incorporated by reference.
- the nucleic acids can be combined with constitutive, tissue-preferred, pathogen- inducible, or other promoters for expression in plants.
- the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
- adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
- in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
- the DNA construct can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues.
- Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase Il (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
- Additional selectable markers include phenotypic markers such as ⁇ -galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 76:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol
- GFP green fluorescent protein
- CYP cyan florescent protein
- selectable marker genes are not meant to be limiting. Any selectable marker gene can be used in the embodiments.
- the gene of the embodiments can be expressed as a transgene in order to make plants resistant to at least one herbicide of the ALS- ⁇ nhibiting, PPO- inhibiting, pigment synthesis-inhibiting, PS ll-inhibiting or synthetic auxin herbicide classes.
- this will allow its expression in a modulated form in different circumstances.
- One can also insert the entire gene, both native promoter and coding sequence, as a transgene.
- using the gene of the embodiments as a transgene will allow quick combination with other traits, such as insect or fungal resistance.
- the nucleic acid sequences of the embodiments can be stacked with any combination of polynucleotide sequences of interest, which may be transgenic or non-transgenic, in order to create plants with a desired phenotype.
- the polynucleotides of the embodiments may be stacked with any other polynucleotides of the embodiments, or with other genes.
- the combinations generated can also include multiple copies of any one of the polynucleotides of interest.
- the polynucleotides of the embodiments can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including and not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Patent No.
- polynucleotides of the embodiments can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxin proteins (U.S. Patent Nos.
- acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations (Lee et al., (1988) EMBO J. 7(5): 1241-1248), resistance to inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene; De Block et al. (1987) EMBO J. 6:2513- 2518); HPPD genes that confer tolerance to HPPD inhibiting herbicides such as mesotrione or isoxaflutole (Matringe et al.
- ALS acetolactate synthase
- high oil e.g., U.S. Patent No. 6,232,529
- modified oils e.g., fatty acid desaturase genes (U.S. Patent No. 5,952,544; WO 94/11516)
- modified starches e.g., ADPG pyrophosphorylase
- PHAs polyhydroxyalkanoates
- agronomic traits such as male sterility (e.g., see U.S. Patent No. 5.583,210), stalk strength, flowering time, yield improvement, or transformation technology traits such as cell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
- stacked combinations can be created by any method including and not limited to cross breeding plants by any conventional or TopCross ® methodology, or genetic transformation.
- the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order.
- a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation.
- the traits can be introduced simultaneously in a co- transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes.
- the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters.
- a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, WO99/25821 , WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.
- Further embodiments include plants obtainable by a method comprising: crossing a plant containing the Nsf1 gene as a first parent plant, with a different plant that lacks an Nsf1 gene as a second parent plant, thereby to obtain progeny comprising the Nsf1 gene of the first parent; and optionally further comprising one or more further breeding steps to obtain progeny of one or more further generations comprising the Nsf1 gene of the first parent.
- Such embodied plants can include both inbred and hybrid plants. Seeds of such plants, including those seeds which are homozygous and heterozygous for the Nsf1 gene, and methods of obtaining plant products resulting from the processing of those seeds are embodied in the invention. Using such seed in food or feed or the production of a corn product, such as flour, meal and oil is also an embodiment of the invention.
- an "ancestral line” or “progenitor” is a parent line used as a source of genes, e.g., for the development of elite lines. "Progeny” are the descendents of the ancestral line, and may be separated from their ancestors by many generations of breeding.
- An "elite line” or “elite variety” is an agronomically superior line or variety that has resulted from many cycles of breeding and selection for superior agronomic performance.
- elite germplasm is an agronomically superior germplasm, typically derived from and/or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line of corn or soybeans.
- Also embodied in the invention is the use of molecular markers to move the gene or transgene into elite lines using breeding techniques.
- Molecular markers can be used in a variety of plant breeding applications (eg see Staub ef a/. (1996) Hortscience 31 : 729-741 ; Ta ⁇ ksley (1983) Plant Molecular Biology Reporter. 1 : 3-8).
- One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS).
- MAS marker-assisted selection
- a molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay, e.g.
- DNA marker assays are less laborious, and take up less physical space, than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line.
- flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed.
- the ideal situation is to have a marker in the gene itself, so that recombination can not occur between the marker and the gene. Such a marker is called a 'perfect marker 1 .
- the nucleic acids of the embodiments may be targeted to the chloroplast for expression.
- the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts.
- transit peptides are known in the art. See, for example, Von Heijne etal. (1991) Plant MoI. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chern. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421 ; and Shah er a/. (1986) Science 233:478-481.
- Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1 ,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho etal. (1996) Plant MoI. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem.
- EPSPS 5-(enolpyruvyl)shikimate-3-phosphate synthase
- the method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue- preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
- the nucleic acids to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831 , herein incorporated by reference.
- the methods of the embodiments may involve, and are not limited to, introducing a polypeptide or polynucleotide into a plant. "Introducing" is intended to mean presenting to the plant the polynucleotide.
- the polynucleotide will be presented in such a manner that the sequence gains access to the interior of a cell of the plant, including its potential insertion into the genome of a plant.
- the methods of the embodiments do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide gains access to the interior of at least one cell of the plant.
- Methods for introducing polynucleotides into plants are known in the art including, and not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
- “Stable transformation” is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof.
- “Transient transformation” or “transient expression” is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
- Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway ef a/. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. ScL USA 83:5602-5606, Agrobacterium- medlated transformation (U.S. Patent Nos. 5,563, 055-and 5,981 ,840), direct gene transfer (Paszkowski et al.
- the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system.
- a site-specific recombination system See, for example, WO99/25821 , WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference.
- the polynucleotide of the embodiments can be contained in transfer cassette flanked by two non-identical recombination sites.
- the transfer cassette is introduced into a plant have stably incorporated into its genome a target site which is flanked by two non-identical recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site.
- the polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
- the cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified.
- the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which maize plant can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like.
- Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species.
- Progeny, variants, and mutants of the regenerated plants are also included within the scope of the embodiments, provided that these parts comprise the introduced polynucleotides.
- the embodiments of the invention may be used to confer or enhance herbicide resistance in plants, especially soy (Glycine max).
- Other plant species may also be of interest in practicing the embodiments of the invention, including, and not limited to, other dicot and monocot crop plants.
- the maize gene of the embodiments is commonly found in the majority of commercial corn lines, most of which are naturally tolerant to at least one, and usually several, synthetic auxin, ALS- , PS II- and pigment synthesis-inhibitor herbicides, such as rimsulfuron, nicosulfuron and mesotrione.
- the present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots.
- plants of interest include, but are not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B.
- plants of interest for the invention include those which have the potential for use as biofuel crops, including, but not limited to, prairie grasses such as switchgrass (Panicum virgat ⁇ m), elephant grass (Pennisetum purpureum), Johnson grass (Sorghum halepense), Miscanthus spp., as well as hybrid poplar and hybrid willow trees.
- prairie grasses such as switchgrass (Panicum virgat ⁇ m), elephant grass (Pennisetum purpureum), Johnson grass (Sorghum halepense), Miscanthus spp., as well as hybrid poplar and hybrid willow trees.
- Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
- tomatoes Locopersicon esculentum
- lettuce e.g., Lactuca sativa
- green beans Phaseolus vulgaris
- lima beans Phaseolus limensis
- peas Lathyrus spp.
- members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
- the embodiments provide not only a gene for use in transgenic applications, but sequences and methods that allow the resistance gene to be used as a marker in corn breeding strategies.
- the gene of the embodiments, or the locus containing it may be identified in a crop line intended to be used for breeding. Breeders would generally want to avoid using crop lines that are sensitive to herbicides where there is usually natural tolerance. Accordingly, the identification of the sequence of the Nsf1 gene will help breeders to identify and avoid creating herbicide-sensitive lines.
- Nucleic acid based markers can be developed and applied using many different technologies. Such technologies include, and are not limited to,
- RFLP Restriction Fragment Length Polymorphism
- SSR Simple Sequence Repeat
- RAPD Random Amplified Polymorphic DNA
- CAS Cleaved Amplified Polymorphic Sequences
- AFLP Amplified Fragment Length Polymorphism
- SNP Single Nucleotide Polymorphism
- SCAR Sequence Characterized Amplified Region
- Sequence Tagged Site (STS) (Onozaki et al., 2004, Euphytica 138:255-262), Single Stranded Conformation Polymorphism (SSCP) (Orita et ai, 1989, Proc Natl Acad Sci USA 86:2766-2770), Inter-Simple Sequence Repeat (ISSR) (Blair et al., 1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon Amplified Polymorphism (IRAP), Retrotransposon-Microsatellite Amplified Polymorphism (REMAP) (Kalendar et al. (1999) Theor. Appl. Genet.
- locus shall refer to a genetically defined region of a chromosome carrying a gene or, possibly, two or more genes so closely linked that genetically they behave as a single locus, responsible for a phenotype.
- a "gene” shall refer to a specific gene within that locus, including its associated regulatory sequences.
- the Nsf1 locus refers to the chromosomal region genetically defined as conferring resistance to at least one herbicide of the ALS- inhibiting, PPO-inhibiting, pigment synthesis-inhibiting, PS ll-inhibiting and synthetic auxin herbicide class.
- One embodiment of the present invention is the isolation of the NsH gene and the demonstration that it is the gene responsible for the phenotype conferred by the presence of the locus. Genetically defined loci are by their nature not as precisely defined in terms of size as genes, which can be delineated molecularly.
- nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The above-defined terms are more fully defined by reference to the specification as a whole.
- Example 1 Identification of the Nsf1 Gene through Positional Cloning
- a BC1 population (expected 50% Nsf1fnsf1, 50% nsf1/nsf1) was developed using the sensitive inbred W703A as the recurrent parent, and either B73 or Q66 as the resistant line. Plants were misted with a 2.3 mM nicosulfuron, 0.5% v/v Kinetic surfactant solution at approximately the V3 stage. Both resistant and sensitive parents were also grown and sprayed as controls. In order to avoid falsely classifying a plant which may have died due to reasons other than the herbicide application, only resistant progeny were sampled and analyzed. A total of 96 resistant plants were used for the initial mapping.
- Q66 and BMS also possess this ORF, although Q66 differs from both B73 and BMS by 3 amino acids ( Figure 2a and 2b)
- Figure 2a and 2b These three variant amino acids are marked with bold type and rectangles in Figures 2a and 2b in the Q66 sequence string to show their positions.
- Analysis of a sensitive line, GA209 shows an insertion of 392 bp relative to the resistant lines which results in a frameshift and an open reading frame of only 338 amino acids (Figure 2b).
- a survey of numerous North American sensitive lines showed that many of the sensitive lines contain this same insertion of unknown DNA.
- Nsf1 is 67% identical to a rice cytochrome P450 which has recently been reported to control sulfonylurea sensitivity in that plant (Accession No: ABC69856, SEQ ID NO: 4).
- Genomic sequence from B73 shows a single intron with the expected GT left border and AG right border. The position of the intron is shown in the sequence listing in SEQ ID NO: 16.
- the cloning of this gene has a number of potential applications. It could be used as a selectable marker for transformation in a sensitive transformable line such as A188 (Ishida ef a/., (1996) Nature Biotechnology 14:745-750).
- a transgene designed to suppress the Nsf1 gene function would function as a dominant negative selectable marker.
- Nsf1 could also be used to create transgenic resistance in other plants, such as soybean, which are sensitive to this subclass of sulfonylureas.
- Vitamin B5 1000X Stock 10.0 g myo-inositol, 0.10 g nicotinic acid, 0.10 g pyridoxine HCI 1 1 g thiamine. Media (per Liter)
- SB196 10 ml_ of each of the above stock solutions, 1 ml_ B5 Vitamin stock, 0.463 g (NH 4 ) 2 SO 4 , 2.83 g KNO 3 , 1 mL 2,4-D stock, 1 g asparagine, 10 g sucrose, pH 5.7.
- SB103 1 pk.
- Murashige & Skoog salts mixture 1 mL B5 Vitamin stock, 750 mg
- SB166 SB103 supplemented with 5 g per liter activated charcoal.
- SB71-4 Gamborg's B5 salts (Gibco-BRL catalog No. 21153-028), 1 mL B5 vitamin stock, 30 g sucrose, 5 g TC agar, pH 5.7.
- Soybean embryogenic suspension cultures were maintained in 35 mL liquid medium (SB196) on a rotary shaker (150 rpm) at 28 °C with fluorescent lights providing a 16-hour day/8-hour night cycle. Cultures were subcultured every 2 weeks by inoculating approximately 35 mg of tissue into 35 mL of fresh liquid media.
- Soybean embryogenic suspension cultures were transformed by particle gun bombardment (see Klein et al. (1987) Nature 327:70-73) using a DuPont Biolistic PDS1000/He instrument.
- the recombinant DNA plasmid used to express Nsf1 was on a separate recombinant DNA plasmid from the selectable marker gene. Both recombinant DNA plasmids were co-precipitated onto gold particles as follows.
- the DNAs in suspension were added to 50 ⁇ l_ of a 20 - 60 mg/mL 0.6 ⁇ m gold particle suspension and then combined with 50 ⁇ L CaCb (2.5 M) and 20 ⁇ l_ spermidine (0.1 M).
- the mixture was pulse vortexed 5 times, spun in a microfuge for 10 seconds, and the supernatant removed.
- the DNA-coated particles are then washed once with 150 ⁇ L of 100% ethanol, pulse vortexed and spun in a microfuge again, and resuspended in 85 ⁇ L of anhydrous ethanol. Five ⁇ L of the DNA-coated gold particles are then loaded on each macrocarrier disk.
- Transformed embryogenic clusters were removed from liquid culture and placed on solid agar medium (SB166) containing no hormones or antibiotics for one week. Embryos were cultured at 26 0 C with mixed fluorescent and incandescent lights on a 16-hour day: 8-hour night schedule. After one week, the cultures were then transferred to SB103 medium and maintained in the same growth conditions for 3 additional weeks. Prior to transfer from liquid culture to solid medium, tissue from selected lines was assayed by PCR for the presence of the chimeric gene. Somatic embryos became suitable for germination after 4 weeks and were then removed from the maturation medium and dried in empty petri dishes for one to five days. The dried embryos were then planted in SB71-4 medium and allowed to germinate under the same light and germination conditions described above. Germinated embryos were transferred to sterile soil and grown to maturity.
- SB166 solid agar medium
- Nsf1 Two different constructs comprising the Nsf1 gene were created to examine herbicide efficacy of the gene when transformed into soybean.
- the Nsf1 constructs were co-bombarded with a 35S:HYG insert to permit event selection using hygromycin.
- One of the two events had significantly better tolerance compared to the controls at 8 DAT and 15 DAT after application of acifluorfen, dicamba, diuron, flumioxazin, isoxaflutole, mesotrione, rimsulfuron, sulcotrione, sulfentrazone, and topramezone treatments.
- the second event had significantly better tolerance compared to the controls at 15 DAT after application of acifluorfen, dicamba, isoxaflutole, mesotrione, rimsulfuron, sulcotrione, sulfentrazone, and topramezone treatments.
- transgenic soybean plants comprising the maize Nsf1 gene displayed better tolerance to a range of different herbicides when compared directly to control plants.
Abstract
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-
2007
- 2007-03-08 US US11/683,737 patent/US20070214515A1/en not_active Abandoned
- 2007-03-09 CN CN2007800165661A patent/CN101437844B/en not_active Expired - Fee Related
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- 2007-03-09 TW TW096108133A patent/TW200808961A/en unknown
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- 2007-03-09 MX MX2008011586A patent/MX2008011586A/en active IP Right Grant
- 2007-03-09 BR BRPI0708711-0A patent/BRPI0708711A2/en not_active Application Discontinuation
- 2007-03-09 WO PCT/US2007/006090 patent/WO2007103567A2/en active Application Filing
- 2007-10-29 US US11/926,180 patent/US20080050824A1/en not_active Abandoned
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Non-Patent Citations (1)
Title |
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See references of WO2007103567A2 * |
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US7838263B2 (en) | 2010-11-23 |
CA2644130C (en) | 2013-05-07 |
EP1991567B1 (en) | 2015-08-19 |
US20090217415A1 (en) | 2009-08-27 |
CN103146828A (en) | 2013-06-12 |
US20070214515A1 (en) | 2007-09-13 |
TW200808961A (en) | 2008-02-16 |
MX2008011586A (en) | 2008-09-22 |
CN101437844B (en) | 2013-04-10 |
CA2644130A1 (en) | 2007-09-13 |
US20080050824A1 (en) | 2008-02-28 |
AR059816A1 (en) | 2008-04-30 |
US20080052798A1 (en) | 2008-02-28 |
CN101437844A (en) | 2009-05-20 |
WO2007103567A2 (en) | 2007-09-13 |
US7705200B2 (en) | 2010-04-27 |
US20080052797A1 (en) | 2008-02-28 |
US20080050823A1 (en) | 2008-02-28 |
US20080050822A1 (en) | 2008-02-28 |
WO2007103567A3 (en) | 2007-10-25 |
US20080052796A1 (en) | 2008-02-28 |
US20080096218A1 (en) | 2008-04-24 |
CN103146828B (en) | 2015-08-19 |
US20080050818A1 (en) | 2008-02-28 |
BRPI0708711A2 (en) | 2011-06-07 |
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